Phosphoryl-Based Peptide Inhibitors of Tyrosine Kinases Growth factor receptors have been strongly linked in a variety of cancers, many of which have are associated with tobacco use such as cancers of the lung, pancreas, larynx, esophagus and bladder. These receptors usually have an intracellular domain that is a protein tyrosine kinase which is capable of catalyzing the transfer of the g-phosphate of ATP to the hydroxyl group of specific tyrosine residues in targeted protein substrates: ATP + Protein (Tyr-OH) -----------> ADP + Protein (Tyr-OPO3-2) The molecular basis for the involvement of these receptors in cancer appears to entail activation of the kinase activity by one of several mechanisms. Some tumors overexpress both the receptor and the growth factor, leading to autocrine regulation of cell growth. Other tumors overexpress only the receptor which can be activated by growth factors produced by other cells (paracrine regulation). Still other tumors express growth factors that can act on surrounding tissues, promoting changes such as angiogenesis that can support tumor growth. A common feature of these three regulatory mechanisms is that they are the result of inappropriate expression of the growth factor receptors and their ligands either at the transcriptional or translational level. That the overexpression of growth factors and receptors is linked to cancer is supported by data indicating that the mitogenic signaling pathways of both the IGF-I receptor and the EGF receptor, for example, can be blocked experimentally by antibodies specific to these receptors, resulting in inhibition of cell growth in tumor cell lines. Most structural studies in receptor kinases have concentrated on the extracellular ligand binding domain, and therefore little is known about the kinase domain of the these receptors. A long-term goal of this project is to develop isoenzyme-specific inhibitors of tyrosine protein kinases. In order to more fully characterize the nature of the involvement of these receptors in cancer and to develop effective, specific peptide-based inhibitors of the target tyrosine protein kinase, we propose the following set of research goals: #1: Express the soluble kinase domain of the human IGF-I and EGF receptors for characterization studies. We propose to express the intracellular b-subunit domain of the IGF-1 receptor via a baculovirus or E. coli expression system and determine the relevant kinetic parameters, i.e., Km and Vmax. An existing baculovirus expression system for the cytoplasmic kinase domain of the analogous EGF receptor will be used for expressing this protein. We will use mass spectrometry to structurally characterize these expressed proteins. #2: Identify and synthesize peptide substrates for the EGF and IGF-1 kinases. We will use innovative multiple peptide synthesis strategies and advanced mass spectrometry to identify good substrates of these kinases. Once identified, these peptide substrate classes will be exploited as the base peptide for constructing bisubstrate analogs. #3: Synthesize phosphotyrosine and adenosine-phosphotyrosine containing bisubstrate analogs as potential selective and potent kinase inhibitors. The peptide portions of these inhibitors will be largely based on the peptide substrate experiments in specific aim #2. #4: Determine the kinetic parameters of the resulting peptide analogs as inhibitors of the EGF and IGF-1 receptor kinase. We will use both cytoplasmic kinase domains of our recombinant IGF-1 and EGF receptors to evaluate our synthetic bisubstrate analogs as inhibitors. A more accurate picture of the chemical mechanism will eventually lead to more rational design of mechanism-based inhibitors.